An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC&#8208;derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl&#8208;2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC&#8208;derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells2016;34:2063&ndash;2078

Mentions:
SQST1/p62, a marker of the UPR and autophagy flux is found in neuronal aggregates in autopsy samples from C9orf72 ALS/FTD cases. We found significantly elevated levels of SQST1/p62 in MNs derived from each C9orf72 patient, and the patient with the highest number of repeats showed up to twofold higher expression of SQST1/p62 compared to control MNs at baseline (p < .05, one‐way ANOVA) (Fig. 6A–6C). No differences were detected in the autophagic marker LC3‐II/LC3‐I in MNs (Supporting Information Fig. S5C, S5D). In CNs carrying the C9orf72 expansions we also found approximately 40% neurons positive for p62 aggregates (Fig. 6D–6F). In contrast to MNs, these results correlated with an increase in levels of LC3‐II in C902‐2 and C902‐10 compared to controls OX3‐9 and AH017‐13 (p < .05, one‐way ANOVA) (Fig. 6G).

Mentions:
SQST1/p62, a marker of the UPR and autophagy flux is found in neuronal aggregates in autopsy samples from C9orf72 ALS/FTD cases. We found significantly elevated levels of SQST1/p62 in MNs derived from each C9orf72 patient, and the patient with the highest number of repeats showed up to twofold higher expression of SQST1/p62 compared to control MNs at baseline (p < .05, one‐way ANOVA) (Fig. 6A–6C). No differences were detected in the autophagic marker LC3‐II/LC3‐I in MNs (Supporting Information Fig. S5C, S5D). In CNs carrying the C9orf72 expansions we also found approximately 40% neurons positive for p62 aggregates (Fig. 6D–6F). In contrast to MNs, these results correlated with an increase in levels of LC3‐II in C902‐2 and C902‐10 compared to controls OX3‐9 and AH017‐13 (p < .05, one‐way ANOVA) (Fig. 6G).

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC&#8208;derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl&#8208;2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC&#8208;derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells2016;34:2063&ndash;2078